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Abstract

Despite more than 30 years of epidermal stem cell research, it is not yet understood how tissue homeostasis is balanced in the interfollicular epidermis (IFE). This is mainly because of the lack of distinct markers to identify human epidermal stem cells. In the hair follicle and the follicle-poor mouse ear epidermis stem cells have been described as slow-cycling cells, also called label-retaining cells (LRCs) (Bickenbach, 1981; Braun et al., 2003; Cotsarelis et al., 1990). It is suggested that these LRCs are responsible for maintaining tissue homeostasis. To address this stem cells need to be identifiable which has yet to be accomplished. Suggesting, that slowly cycling is a characteristic also of epidermal stem cells in the IFE, the aim of this study was the isolation and characterization of LRCs. Since investigation on human skin is restricted, we employed a human in vitro 3D organotypic skin model (Muffler et al., 2008). For the detection of LRCs we utilized an inducible Tet-Off-H2B-GFP lentiviral reporter vector. Thus, a method was developed to successfully infect normal human adult keratinocytes (NHEK) with the lentiviral vector and these keratinocytes were further propagated in organotypic cultures (OTC) to establish long-term skin equivalents. In pulse-chase approaches (2 weeks pulse, 6 weeks chase), viable LRCs of the IFE could be functionally identified as slow-cycling cells and isolated as GFP positive cells from OTCs. To further characterize these cells, microarray analysis was performed of the isolated basal LRCs and their respective non-labeled basal cell fraction. This analysis revealed 102 genes to be differentially regulated in the LRCs. We could show that a high fraction of upregulated genes in LRCs were ECM or ECM remodeling genes which could be verified by qRT-PCR. Further characterization on protein level is in progress. From this profile it is suggested, that LRCs actively modulate their local microenvironment, referred to as niche, by secreting unique ECM components. This distinct ECM may well serve for enhanced adhesion of LRCs to the niche as well as a reservoir for growth factors regulating cell signaling. Ingenuity pathway analysis further suggested a role for TGFβ signaling in this regulation. Accordingly, stimulation experiments provided first evidence that TGFβ-1 stimulates upregulation of those genes in NHEKs in 2D cultures that were highly upregulated in LRCs in their tissue context. Thus, we propose that LRCs exhibit a distinct molecular profile that compared to all other basal cells allows them to establish their specific regulatory niche. Their functional role as epidermal stem cells has now to await further investigations.